Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
  • C07C237/30—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having the nitrogen atom of the carboxamide group bound to hydrogen atoms or to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/185—Saturated compounds having only one carboxyl group and containing keto groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2603/00—Systems containing at least three condensed rings
  • C07C2603/02—Ortho- or ortho- and peri-condensed systems
  • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
  • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
  • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
  • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
  • C07C2603/18—Fluorenes; Hydrogenated fluorenes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention is directed to a new class of lipids, more specifically ether-lipids having a polar headgroup, and vesicles comprising these lipids, methods of their preparation as well as their uses in medical applications.
• Molecular recognition such as between receptor ligand, antigen-antibody, DNA-protein, sugar-lectin, RNA-ribosome, etc. is an important principle underlying many biological systems and is being applied to many artificially created biological systems for use in medical applications, such as in artificial (micro- or nano-) particulate systems including polymeric beads, vesicular lipids, microemulsions, and the like.
• a molecular recognition based application is the use of targeted delivery of diagnostic or therapeutic compounds, such as antiviral, chemotherapeutic or imaging agents, to specific sites, which allows to overcome the limitations associated with nonspecific delivery (such as in vivo clearance time, potential toxicity, problems associated with membrane transport of an agent and the like) and thus greatly increases their effectiveness.
• Various recognition-based strategies have been used to improve the delivery of compounds into the intracellular environment (i.e. to specific cell compartments) of a target cell to exert its biological activity, in particular delivery through specific transporters involving the use of biological or artificial carriers, such as viral vectors, cationic polymers, such as polylysine, polyarginine and the like (see, e.g. WO 79/00515, WO 98/52614), lipid carriers, and various other conjugate systems.
• lipid vesicles as artificial carriers, e.g. liposomes, micelles, nanoparticles, which have been extensively developed and analyzed as drug delivery vehicles due to their ability to reduce systemic exposure of a biologically active agent, thereby overcoming problems associated with degradation, solubility, etc. and providing an increase in blood circulation times.
• Actively targeted delivery of a biologically active agent involves derivatizing the lipids of the lipid vesicle (either prior or after vesicle formation) with a targeting ligand that serves to direct (or target) the vesicle to specific cell types such as cancer cells or cells specific to particular tissues and organs, such as hepatocytes, after in vivo administration (see, for example, U.S. Pat. No. 6,316,024 and U.S. Pat. No. 6,214,388; Allen et al., Biochim. Biophys. Acta, 1237:99-108 (1995); Blume et al., Biochim. Biophys. Acta, 1149:180-184 (1993)).
• receptors that are overexpressed in specific cell types, which include for example folic acid receptor (overexpressed in a variety of neoplastic tissues, including breast, ovarian, cervical, colorectal, renal, and nasoparyngeal tumors), epidermal growth factor receptor (EGFR) (overexpressed in anaplastic thyroid cancer and breast and lung tumors), metastin receptor (overexpressed in papillary thyroid cancer), ErbB family receptor tyrosine kinases (overexpressed in a significant subset of breast cancers), human epidermal growth factor receptor-2 (Her2/neu) (overexpressed in breast cancers), tyrosine kinase-18-receptor (c-Kit) (overexpressed in sarcomatoid renal carcinomas), HGF receptor c-Met (overexpressed in esophageal adenocarcinoma), CXCR4 and CCR7 (overexpressed in breast cancer), endothelin-A receptor
• Any agent which selectively binds to such a specific receptor cell or tissue to be treated or assayed may be attached to a lipid vesicle and act as a targeting or receptor ligand.
• targeting ligands have been attached to a lipid or lipid vesicle surface through a long chain (e.g. polymeric) linker.
• folic acid based conjugates have been used to provide a targeted delivery approach of a therapeutic compound useful for the treatment and/or diagnosis of a disease, allowing a reduction in the required dose of therapeutic compounds (see e.g. WO 02/094185, U.S. Pat. No. 6,335,434, WO 99/66063, U.S. Pat. No. 5,416,016).
• galactose- and galactosamine-based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as HBV and HCV infection or hepatocellular carcinoma while allowing a reduction in the required dose of therapeutic compounds required for treatment (see e.g. U.S. Pat. No. 6,030,954).
• antigen display systems which involve presentation of both “self” and “foreign” proteins (antigens) to the immune system to generate T cell activation, modulation or tolerance.
• the receptor ligand interactions in antigen-presenting systems that contribute to the desired immune response or absence thereof are complex and difficult to assess, being influenced by various parameters such as ligand densities, presence of coreceptors, receptor ligand affinities and surface conditions.
• live cell based systems may be optimal for mimicking cell-cell interaction to achieve the desired induction of tolerance or immune response, they are dependent on a regulated expression of the surface molecules including possibly expression of additional “costimulatory” and/or adhesion molecules on its surface membrane at a sufficient therapeutic level.
• known artificial systems range from genetically engineered subcellular antigen presenting vesicles, which carry the molecules required for antigen presentation and T-lymphocyte activation or inhibition on their surface (WO 03/039594) to systems on the basis of cell-sized, biodegradable microspheres based, antigen presenting system (WO 07/087,341).
• the present application provides a new class of lipids and vesicles comprising these lipids for use as a carrier or display system, which allows overcoming the limitations described above.
• the present invention is directed to a new class of lipids and vesicles comprising these lipids for use in various medical applications. More specifically the present invention is directed to ether-lipid compounds that are characterized by at least two ether-linked hydrocarbon chains and a headgroup comprising a short, straight-chain amino acid having up to 6 carbon atoms, in free, protected or activated form or optionally derivatized with at least one spacer group.
• the present invention relates to a compound of general formula I
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• L is a group of formula (a)
• R 1 represent H or a group of formula —(CH 2 ) 2 —OR b1 , R 1′ , represent H or a group of formula —(CH 2 ) 2 —OR b2 , R 2 represents H or a group of formula —CH 2 —OR c , R 2′ represents H or a group of formula —OR d or —CH 2 —OR d , R 3 represents H or a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e , R a , R b1 , R b2 , R c , R d , R e represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain, m is 1, 2 or 3, with the proviso that at least one of R 1 , R 1′ , R 2 , R 2′ , R 3 is not H.
• the compounds of the invention include all possible stereoisomers of the compounds, such as geometric isomers, e.g. Z and E isomers (cis and trans isomers), and optical isomers, e.g. diastereomers and enantiomers, in either pure form or in mixtures thereof.
• the invention is directed towards non-derivatized lipid compounds, wherein neither P 1 , P 2 , P 3 is a spacer group.
• the non-derivatized lipid compounds include (i) lipid compounds in free form, wherein neither of P 1 , P 2 , P 3 is an activating or protecting group, (ii) protected lipid compounds, wherein at least one of P 1 , P 2 , P 3 is a protecting group, and (iii) activated lipid compounds, wherein P 1 is an activating group.
• the invention is directed towards lipid-spacer derivatives, wherein at least one of P 1 , P 2 , P 3 is a spacer group.
• the compounds of the present invention comprise all possible permutations of groups R 1 , R 1′ , R 2 , R 2′ , R 3 and the substructures R a , R b1 , R b2 , R c , R d , R e thereof.
• group R 3 is H. More specifically, either (i) R 3 is H and both R 1 and R 1′ are H, or (ii) R 3 is H and both R 2 and R 2′ are H.
• the invention is directed towards compounds of formula Ia,
• the invention is directed towards compounds of formula Ia, wherein L is a group of formulas (b) or (c)
• R 1 , R 1′ , R 2 , R 2′ are H and R 3 is either a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e .
• the invention is directed towards compounds of formula Ib,
• R 3 is a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e
• P 1 , P 2 , P 3 , Y, R a , R e and m are defined as above for a group of formula I.
• the present invention is directed towards compositions in the form of vesicles (present compositions), e.g. liposomes, micelles, nanoparticles and the like.
• the vesicles of the invention comprise at least one compound of the invention or a mixture of various compounds of the invention, optionally in admixture with one or more other vesicle-forming compounds.
• the present invention is directed towards a method for preparing a compound and composition of the invention.
• kits comprising a compound or a composition of the invention, preferably in lyophilized form.
• the compounds and compositions of the present invention find use as a delivery vehicle e.g. for the targeted delivery of one or more bioactive agents or for use in an antigen display system.
• This aspect of the present compounds and compositions is part of an international application filed concurrently, which is incorporated herein in its entirety.
• compound refers to a compound of the invention, which comprise a linear, bifunctional amino acid at the head group, more specifically a 2-amino-alkanedioic acid (having up to six carbon atoms), such as aspartic acid, glutamic acid, and the like.
• the compounds of the invention include both “non-derivatized (lipid) compounds”, which are either in free form (“free (lipid) compound”), in protected form (“protected (lipid) compound”) or in activated form (“activated (lipid) compound”) and thus carry no covalently attached spacer groups, as well as “derivatized (lipid) compounds” (or “lipid spacer derivative”), which are conjugates of non-derivatized (lipid) compounds with one or more spacer groups.
• a protected or activated (lipid) compound refers to a compound of the invention which has been modified site-specifically to contain a protecting or activating group, respectively.
• the modification takes place at the head group, more specifically at the reactive sites of the amino acid, more preferably at the N- and/or Y-groups with suitable protecting or activating groups (e.g. in form of P 1 , P 2 , P 3 ), respectively, known in the art.
• a “lipid spacer derivative” refers to a compound of the invention which has been modified site-specifically to contain a spacer group. The modification takes place at the head group, more specifically at the reactive sites of the amino acid, more preferably at the N- and/or Y-group (or CO-group if Y is a covalent bond) with suitable spacer groups (e.g. in form of P 1 , P 2 , P 3 ) known in the art using known coupling techniques.
• suitable spacer groups e.g. in form of P 1 , P 2 , P 3
• composition refers to a composition which comprises at least one compound of the invention.
• exemplary compositions include vesicles or vesicular compositions, which in their broadest interpretation include any association of at least one lipid compound of the invention with other materials and structures.
• suitable vesicular compositions include, but are not limited to, liposomes, micelles, microspheres, nanoparticles and the like.
• a vesicular composition refers to a spherical entity having an internal void or a solid core.
• Vesicles may be formulated from synthetic or naturally-occurring lipids, including one or more compounds of the present invention, and mixtures thereof.
• the lipids may be in the form of a monolayer or a bilayer. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric.
• the lipid vesicles include such entities commonly referred to as liposomes (i.e. a vesicle including one or more concentrically ordered lipid bilayer(s) with an internal void), micelles (i.e. a vesicle including a single lipid monolayer with an internal void), nanospheres, and the like.
• the lipids may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of about two or about three monolayers or bilayers) or a multilamellar vesicle (comprised of more than about three monolayers or bilayers).
• they may be used to coat a preexisting vesicle such as a nanoparticle, e.g. a nanosphere.
• An internal void of the vesicles may be filled with a liquid, including, for example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid material, including, for example, one or more biologically active agents.
• a vesicular composition refers to compositions in form of clusters, tubes and the like.
• compositions of the invention may comprise one or more biologically active agents, which are either embedded or enclosed therein or attached thereto (covalently and non-covalently). More specifically, the vesicular compositions of the invention may comprise in the internal void one or more biologically active agents (for delivery functions) and/or may be derivatized at their surface with one or more biologically active agents (for either targeting or display functions). This is part of an application filed concurrently, which is incorporated herein in its entirety.
• co-lipid or “vesicle-forming (co-)lipid” as used herein refers to lipids which may optionally be present as additional lipids in the lipid compositions of the invention and may include acyclic and cyclic, saturated or unsaturated lipids of natural or synthetic origin.
• a co-lipid may be a neutral lipid, a cationic lipid or an anionic lipid.
• a cationic lipid has a positive net charge and may include lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, e.g.
• DOTAP methylsulfate
• DDAB dimethyldioctadecyl ammonium bromide
• 1,2-diacyloxy-3-trimethylammonium propanes (including but not limited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and distearoyl; also two different acyl chain can be linked to the glycerol backbone); N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes, (including but not limited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and distearoyl; also two different acyl chain can be linked to the glycerol backbone); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
• Chem. 1994, 269, 2550-2561 such as: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), 1,2-disteryloxypropyl
• cationic triesters of phospahtidylcholine i.e. 1,2-diacyl-sn-glycerol-3-ethylphosphocholines, where the hydrocarbon chains can be saturated or unsaturated and branched or non-branched with a chain length from C 12 to C 24 , the two acyl chains being not necessarily identical.
• Neutral or anionic lipids have a neutral or anionic net charge, respectively. These can be selected from sterols or lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids with a neutral or negative net change.
• Useful neutral and anionic lipids thereby include: phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to a specific sugar), fatty acids, sterols, containing a carboxylic acid group for example, cholesterol, cholesterol sulfate and cholesterol hemisuccinate, 1,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, DOPE, 1,2-diacyl-glycero-3-phosphocholines and sphingomyelin.
• the fatty acids linked to the glycerol backbone are not limited to a specific length or number of double bonds.
• Phospholipids may also have two different fatty acids.
• the present invention is directed to a new class of lipids and vesicles comprising these lipids for use in various medical applications. More specifically the present invention is directed to ether-lipid compounds H-L, wherein L is a lipidic group comprising at least two ether-linked hydrocarbon chains and H is a headgroup comprising a short, straight-chain amino acid ( ⁇ -amino acid) having up to 6 carbon atoms and derivatives thereof.
• the present invention relates to a compound of general formula I
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• L is a group of formula (a)
• R 1 represent H or a group of formula —(CH 2 ) 2 —OR b1 , R 1′ , represent H or a group of formula —(CH 2 ) 2 —OR b2 , R 2 represents H or a group of formula —CH 2 —OR c , R 2′ represents H or a group of formula —OR d or —CH 2 —OR d , R 3 represents H or a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e , R a , R b1 , R b2 , R c , R d , R e represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain, m is 1, 2 or 3, with the proviso that at least one of R 1 , R 1′ , R 2 , R 2′ , R 3 is not H.
• group R 3 is H. More specifically, either (i) R 3 is H and R 1 and R 1′ are H, or (ii) R 3 is H and R 2 and R 2′ are H.
• the invention is directed towards compounds of formula Ia, wherein L is a group of formulas (b) or (c)
• R 2 is H and R 2′ is —OR d or —CH 2 —OR d .
• R 2 is —CH 2 —OR c and R 2′ is —OR d or R 2′ is —CH 2 —OR d .
• the invention is preferably directed to compounds of formula Ia, wherein L is a group of formula (b1), (b2), (b3) or (b4):
• R a , R c and R d are independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain.
• one of R 1 and R 1′ is H. In another preferred embodiment of group (c) neither of R 1 and R 1′ is H.
• the invention is preferably also directed to compounds wherein L is a group of formula (c1) or (c2):
• R a , R b1 , R b2 are defined as above.
• groups R 1 , R 1′ , R 2 , R 2′ are H and R 3 is either a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e .
• R 3 is a group of formula —(CH 2 ) 2 —OR e or —(CH 2 ) 3 —OR e
• P 1 , P 2 , P 3 , Y, R a , R e and m are defined as above for a group of formula I.
• Preferred embodiments of the invention are thus compounds of formula I (or formula Ia) represented by compounds of formulas II or III
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• R 1 represents H or a group of formula —(CH 2 ) 2 —OR b1
• R 1′ represent H or a group of formula —(CH 2 ) 2 —OR b2
• R 2 represents H or a group of formula —CH 2 —OR c
• R 2′ represents H or a group of formula —OR d or —CH 2 —OR d
• R a , R b1 , R b2 , R c , R d represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain
• m is 1, 2 or 3, with the pro
• compounds of formula II are compounds of formula IIa, IIb, IIc or IId,
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• R a , R c , R d represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain
• m is 1, 2 or 3.
• Y represents O, N, S or a covalent linkage
• P 1 represents H, a Y-protecting group or a Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• R a , R b1 , R b2 represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain
• m is 1, 2 or 3.
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• R a , R e represent independently of each other a saturated or unsaturated, straight or branched hydrocarbon chain
• m is 1, 2 or 3.
• the compounds of the present invention contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers, e.g. Z/E isomers or cis/trans isomers), enantiomers or diastereomers.
• stereoisomers such as double-bond isomers (i.e., geometric isomers, e.g. Z/E isomers or cis/trans isomers), enantiomers or diastereomers.
• the chemical structures depicted herein encompass all possible configurations at those chiral centers including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) the enriched form (e.g., geometrically enriched, enantiomerically enriched or diastereomerically enriched) and enantiomeric and stereoisomeric mixtures.
• the individual isomers may be obtained using the corresponding isomeric forms of the starting material.
• enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan.
• the compounds of the invention described herein may also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
• saturated or unsaturated, straight or branched hydrocarbon chain refers to a saturated or unsaturated, straight or branched hydrocarbon chain having 6 to 30, preferably 10 to 22 carbon atoms.
• saturated in combination with hydrocarbon chain refers to a straight or branched alkyl chain, containing 6 to 30, preferably 10 to 22 carbon atoms.
• examples include, but are not limited to, capryl (decyl), undecyl, lauryl (dodedecyl), myristyl (tetradecyl), cetyl (hexadecyl), stearyl (octadecyl), nonadecyl, arachidyl (eicosyl), heneicosyl, behenyl (docosyl), tricosyl, tetracosyl, pentacosyl, including branched isomers thereof, e.g.
• the term “unsaturated” in combination with hydrocarbon chain indicates that fewer than the maximum possible number of hydrogen atoms are bonded to each carbon in the chain giving rise to one or more carbon-carbon double or triple bonds.
• the number of unsaturated bond(s) in an unsaturated hydrocarbon chain is 1, 2, 3 or 4, preferably 1 or 2.
• alkenyl groups include, but are not limited to, monounsaturated alkenyls, such as decenyl, undecenyl, dodecenyl, palmitoleyl, heptadecenyl, octadecenyl (elaidyl, oleyl, ricinolenyl), nonadecenyl, eicosenyl, heneicosenyl, docosenyl (erucyl), tricosenyl, tetracosenyl, pentacosenyl, and the branched chain isomers thereof, as well as polyunsaturated alkenyls such as octadec-9,12-dienyl (linoleyl, elaidolinoleyl), octadec-9,12,15-trienyl (linolenyl, elaidolinolenyl), 9(Z
• alkynyl groups include, but are not limited to hexadec-7-ynyl and octadec-9-ynyl.
• branched in combination with hydrocarbon refers to a hydrocarbon chain having a linear series of carbon atoms as a main chain with at least one substituent of one or more carbon atoms as subordinate chain (or branching groups).
• subordinate chains include one or more (C1-6)alkyl groups, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl group, tert-butyl, pentyl, hexyl and the like, one or more (C1-6)alkenyl groups, such as vinyl, allyl, propenyl, isopropenyl, 2-butenyl and the like, or one or more (C1-6)alkynyl groups, such as ethynyl, propynyl, butynyl and the like.
• Preferred subordinate chains are (C1-6)alkyl groups, most preferred methyl and ethyl.
• the compounds of the invention comprise preferably at least two hydrocarbon chains, preferably 2, 3, 4, 5 or 6 hydrocarbon chains, most preferably 2 or 3 hydrocarbon chains, wherein the main chain of the hydrocarbon chains are the same or different, preferably the same, and are selected from an alkyl chain, an alkenyl chain, and an alkynyl chain, preferably an alkyl and an alkenyl chain.
• the compounds of the invention carry two alkyl chains, which can be the same or different, preferably the same.
• the hydrocarbon chains R a , R b1 , R b2 , R c , R d , R e are preferably selected from myristyl, palmityl, stearyl, oleyl, linoleyl and phytanoyl.
• alkyl alkoxy
• alkenyl alkynyl
• alkyl refers to a straight or branched alkylchain, containing 1 to 12, preferably 1 to 8 carbon atoms.
• alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.
• alkoxy refers to an —O-alkyl radical. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, and butoxy.
• alkenyl refers to a straight or branched unsaturated alkyl group having one or more carbon-carbon double bonds.
• alkyl, alkenyl, and alkoxy groups may be optionally substituted with further groups.
• substituents include, but are not limited to, halo, hydroxyl, amino, cyano, nitro, mercapto, alkoxycarbonyl, amido, carboxy, alkylsulfonyl, alkylcarbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfonamido, aryl, heteroaryl, cyclyl, and heterocyclyl.
• aryl refers to an aromatic carbocyclic radical containing about 6 to about 10, preferably 5 to 7 carbon atoms.
• the aryl group may be optionally substituted with one or more aryl group substituents which may be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene and —NRR′, wherein R and R′ are each independently hydrogen, alkyl, aryl and aralkyl.
• heteroaryl refers to an aryl moiety as defined above having at least one heteroatom (e.g., N, O, or S).
• heteroaryl moiety include furyl, furylene, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl, isoquinolyl and indolyl.
• (hetero)aryloxy refers to an (hetero)aryl-O-group wherein the (hetero)aryl group is as previously described.
• exemplary aryloxy groups include phenoxy and naphthoxy.
• (hetero)aralkyl refers to an (hetero)aryl-alkyl-group wherein (hetero)aryl and alkyl are as previously described.
• Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
• (hetero)aralkyloxy refers to an (hetero)aralkyl-O-group wherein the (hetero)aralkyl group is as previously described.
• An exemplary aralkyloxy group is benzyloxy.
• cycloalkyl refers to a saturated or unsaturated, non-aromatic, cyclic hydrocarbon moiety having 6 to 10 carbon atoms, such as cyclohexyl or cyclohexen-3-yl.
• heterocycloalkyl refers to a cycloalkyl as defined herein having at least one ring heteroatom (e.g., N, O, or S), such as 4-tetrahydropyranyl or 4-pyranyl.
• Aryl, heteroaryl, cycloalkyl, heterocycloalkyl as mentioned herein include both substituted and unsubstituted moieties, unless specified otherwise.
• Possible substituents on cycloalkyl, heterocycloalkyl, aryl, and heteroaryl include (C1-C10)alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl, (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (C1-C10)alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, (C 1 -C 10 )alkylamino, (C1-C20)dialkylamino, arylamino, diarylamino, hydroxyl, halogen, thio, (C1-C10)alkylthio, arylthio, (C1-C10)alkylsulfonyl, arylsulfonyl
• Group Y is O, N, S or a covalent linkage, preferably O or N, most preferably N. It is understood that if group Y is a covalent linkage, —S 1 —X 1 is directly linked to the CO-group.
• a “protecting group” is a moiety that can be selectively attached to and removed from a particular chemically reactive functional group in a molecule to prevent it from participating in undesired chemical reactions.
• the protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule.
• protecting group if used in relation to a N-group (such as P 1 with Y being N, P 2 or P 3 ) in one of the compounds is an amino-protecting group, if used in relation to a COO-group (such as P 1 with Y being O) in one of the compounds is a carboxyl-protecting group, if used in relation to a CO-group (such as P 1 with Y being a covalent linkage) in one of the compounds is a carbonyl-protecting group, and if used in relation to a S-group (such as P 1 with Y being S) in one of the compounds is a sulfur-protecting group.
• protecting groups for various functional groups such as e.g. carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like, are well-known to those skilled in the art and are described, for example, in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, N.Y., 1999, and references cited therein.
• a “carboxyl-protecting group” includes but is not limited to benzhydryl, benzyl esters, such as benzyl, and o- or p-nitrobenzyl, p-methoxybenzyl, alkyl esters, such as methyl, t-butyl, 4-pyridylmethyl 2-naphthylmethyl, 2,2-trichloroethyl, silyl esters, such as 2-trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, 2-(trimethylsilyl)ethyl; orthoesters, such as trimethyl- or triethyl orthoacetate; oxazoline, allyl, 2-chloroallyl, phenacyl, acetonyl, p-methoxyphenyl.
• Preferred groups include benzyl, t-butyl, 4-pyridylmethyl 2-naphthylmethyl
• An amide protecting group (e.g. group P 1 with Y being N) includes but is not limited to a phthalimide or a trifluoroacetamide protecting group.
• amino-protecting group includes both acyclic as well as cyclic protecting groups (P 2 and P 3 ), for example each of groups P 2 and P 3 may represent a protecting group which can be the same or different or P 2 and P 3 form together with the N to which they are bound a cyclic protecting group.
• Typical groups include, but are not limited to, carbamates, such as Boc (t-butyloxycarbonyl, Cbz (carboxybenzyl), Fmoc (fluorenylmethyloxycarbonyl), alloc (allyloxycarbonyl), methyl and ethyl carbamates; trityl, benzyl, benzylidene, tosyl and the like; cyclic imide derivatives, such as succinimide and phthalimide; amides, such as formyl, (un)substituted acetyl, and benzoyl; and trialkyl silyl groups, such as t-butyldimethylsilyl and triisopropylsilyl.
• Preferred amino-protecting groups include Boc, Cbz, Fmoc, benzyl, acetyl, benzoyl, trityl and the like.
• activated or “activating,” for example, as used in connection with any of the terms “group”, “amine group” “carboxyl group”, “spacer group”, refer to a chemical moiety that render a chemical functionality more sensitive to modification under certain reaction conditions such that the activated chemical functionality can react under appropriate conditions with a second chemical group thereby forming a covalent bond.
• an activating group may convert a poor leaving group into a good leaving group or increase (or decrease) susceptibility to nucleophilic attack or other chemical transformations.
• a “carboxyl activating group” is meant to refer to a moiety that replaces the hydrogen or hydroxyl of a carboxyl group, thereby altering the chemical and electronic properties of the carboxyl group such that the carboxyl group is more susceptible to nucleophilic attack or substitution.
• exemplary carboxyl activating groups include, for example, alkyl, aryl, aralkyl, heteroaryl, heterocyclyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heterocyclylcarbonyl, C(S)O-aryl, C(S)O-alkyl, silyl or substituted alkylcarbonyl.
• An example of an aryl carboxyl activating group is pentahalophenyl, such as pentafluorophenyl, and an example of an alkylcarbonyl carboxyl activating group is acetyl or trifluoroacetyl.
• the carboxyl activating groups may be optionally substituted.
• An example of a substituted carboxyl activating group is substituted alkylcarbonyl, for example, carboxyl substituted alkylcarbonyl, such as succinyl (3-carboxylpropionyl).
• Carboxyl activation in which the hydrogen of the —C( ⁇ O)—OH group is replaced may also involve the use of coupling agents, which are moieties that promote nucleophilic addition reactions, i.e. substituents which have a net electron withdrawing effect on the carbonyl.
• Such groups act to assist or promote the coupling of, or to improve the rate of the coupling of, carboxylate groups with compounds having reactive functionalities, for example, nucleophiles, including amino groups such as in the formation of amido functionality.
• Coupling agents are well known to one ordinarily skilled in the art and are described, for example, in Larock, R. C., Comprehensive Organic Transformations, VCH Publishers, Inc., NY (1989), and Carey, F. A., and Sundberg, R. J., Advanced Organic Chemistry, 3 rd Edition, Plenum Press, NY (1990), the disclosures of each of which are hereby incorporated herein by reference in their entireties.
• Carboxyl activation in which the hydroxyl group of the —C( ⁇ O)—OH group is replaced includes e.g. replacing the hydroxyl by a moiety such as a halo group, such as fluoro, chloro, bromo or iodo, giving a carboxylic acid halide, which is more susceptible to nucleophilic attack or substitution.
• a halo group such as fluoro, chloro, bromo or iodo
• typical activating or coupling groups include, but are not limited to, esters and amides such as hydroxybenzotriazole, imidazole, a nitrophenol, pentachlorophenol, N-hydroxysuccinimide, dicyclohexylcarbodiimide, N-hydroxy-N-methoxyamine, and the like; acid anhydrides such as acetic, formic, sulfonic, methanesulfonic, ethanesulfonic, benzenesulfonic, or p-tolylsulfonic acid anhydride, and the like; and acid halides such as the acid chloride, bromide, or iodide.
• esters and amides such as hydroxybenzotriazole, imidazole, a nitrophenol, pentachlorophenol, N-hydroxysuccinimide, dicyclohexylcarbodiimide, N-hydroxy-N-methoxyamine, and the like
• acid anhydrides such as
• the activated carbonyl compound is obtained by reacting a reactive moiety of choice with the carbonyl compound using standard procedures.
• the activated carbonyl compound may be generated in situ, or may be provided in isolated form, as appropriate.
• Exemplary reactive moieties to obtain the activated compounds cited above include the respective groups containing isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halo substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)proprionamide, glyoxal and aldehyde.
• spacer or “spacer group” in conjunction with groups P 1 , P 2 , P 3 is used herein to refer to a bivalent branched or unbranched chemical group which allows to link the compound of the invention to a further moiety, i.e. a bioactive group in sufficient distance to eliminate any undesired interaction between compound and further moiety and/or to reduce any steric hindrance (caused by the compound itself or any other neighbouring molecules) that may impact the biological activity of the further moiety (such as affinity binding of ligands to their receptor).
• the spacer groups may be of different length and may be (hydrolytically, enzymatically and chemically) stable or may include a cleavable linkage.
• Cleavable linkages of the invention may be selected to be cleaved via any form of cleavable chemistry, e.g. chemical, enzymatic, hydrolytic and the like.
• cleavable linkers include, but are not limited to, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers, ultrasound-sensitive linkers, x-ray cleavable linkers, etc.
• Groups P 1 , P 2 , P 3 may represent independently of each other H, a protecting group or a spacer group. More specifically P 1 represents H, a Y-protecting group or a Y-activating group or a spacer group S 1 ; P 2 represents H, an amino-protecting group or a spacer group S 2 ; and P 3 represents H, an amino-protecting group or a spacer group S 3 ; or P 2 and P 3 form together with the N to which they are bound a ring structure.
• spacers may or may not be end-group activated to allow for linkage of the spacer modified compound of the invention to a further moiety, such as bioactive group.
• a “spacer group” (also termed groups S 1 , S 2 , S 3 ) represents a short spacer group or a long-chain spacer group selected from an alkylene chain optionally comprising one or more of the groups selected from ketone, ester, ether, amino, amide, amidine, imide, carbamate or thiocarbamate functions, glycerol, urea, thiourea, double bonds or aromatic rings.
• a short spacer group (or groups S 1 , S 2 , S 3 ) may be chosen from (C1-C12)alkyl, (C2-C12)alkenyl, aryl, aralkyl, heteroaryl.
• a long-chain spacer group (or groups S 1 , S 2 , S 3 ) may be chosen from polymeric radicals of formula —W—(CH 2 —) k —W′—, wherein k is an integer between 13 and 3000, and W and W′ are reactive groups able to react with amino, carboxyl, hydroxy or thio groups and wherein one or more of the non-adjacent CH 2 groups may independently be replaced by aryl, heteroaryl, —CH ⁇ CH—, —C ⁇ C—, or a hydrophilic (or polar) group selected from —O—, —CO—, —CO—O—, —O—CO—, —NR′—, —NR′—CO—, —CO—NR′—, —NR′—CO—O—, —O—CO—NR′—, —NR′—CO—NR′—, and —O—CO—O—, wherein R′ represents hydrogen or (C1-C12)alkyl. It is
• Preferred spacer groups include hydrophilic polymeric radicals (with an increased affinity for aqueous solutions), i.e. polymers containing repeating structural units that comprise one or more of the above hydrophilic (or polar) groups in their alkylene backbone.
• hydrophilic polymeric radicals include polyoxy(C 2 -C 3 )alkylenes (e.g. polyethylene glycol (PEG) or polypropylene glycol (PPG)), polysaccharides (e.g. dextran, pullulan, chitosan, hyaluronic acid), polyamides (e.g.
• polyamino acids semisynthetic peptides and polynucleotides
• polysialic acid polyesters (e.g. polylactide (PLA), polylactid-co-glycolid (PLGA)), polycarbonates, polyethyleneimines (PEI), polyimides, polyvinyl acetate (PVA).
• a preferred spacer is “PEG” or “polyethylene glycol”, which encompasses any water-soluble poly(ethylene oxide).
• PEG means a polymer that contains a majority, e.g. >50%, of subunits that are —CH 2 CH 2 O—.
• Different forms of PEG may differ in molecular weights, structures or geometries (e.g., branched, linear, forked PEGs, multifunctional, and the like).
• PEGs for use in the present invention may preferably comprise one of the two following structures: “—O(CH 2 CH 2 O) m —” or “—CH 2 CH 2 (CH 2 CH 2 O) m —CH 2 CH 2 —,” where m is 3 to 3000, and the terminal groups and architecture of the overall PEG may vary. As indicated above, depending on its use, PEG may be in end-capped form.
• the end capping group is generally a carbon-containing group typically comprised of 1-20 carbons and is preferably alkyl (e.g., methyl, ethyl or benzyl) although saturated and unsaturated forms thereof, as well as aryl, heteroaryl, cyclyl, heterocyclyl, and substituted forms of any of the foregoing are also envisioned.
• the end capping group is generally a carbon-containing group typically comprised of 1-20 carbon atoms and an oxygen atom that is covalently bonded to the group and is available for covalently bonding to one terminus of the PEG.
• the group is typically alkoxy (e.g., methoxy, ethoxy or benzyloxy) and with respect to the carbon-containing group can optionally be saturated and unsaturated, as well as aryl, heteroaryl, cyclyl, heterocyclyl, and substituted forms of any of the foregoing.
• non-end-capped terminus is typically a hydroxyl, amine or an activated group that can be subjected to further chemical modification when PEG is defined as “—CH 2 CH 2 CH 2 (CH 2 CH 2 O) m —CH 2 CH 2 —”
• the end-capping group can also be a silane.
• the invention is directed to compounds of the following formulas V, VI and VII:
• Y represents O, N, S or a covalent linkage
• P 1 represents H, an Y-protecting group or an Y-activating group or a spacer group
• P 2 , P 3 represent independently of each other H, an amino-protecting group or a spacer group, or P 2 and P 3 form together with the N to which they are bound a ring structure
• p1, q1, p2, q2, p3, q3 are independently of each other 1 to 23, with the proviso that the sum of p1 and q1, p2 and q2, p3 and q3 is from 12 to 24.
• the present invention is directed towards a method for preparing a compound of the invention.
• the compounds of the invention are particularly suitable for use in the preparation of vesicular compositions, such as liposomes, micelles and (lipid coated) nanoparticles.
• the present invention is directed towards vesicular compositions which are composed of at least one compound of the invention.
• vesicles comprise non-derivatized compounds or derivatized compounds comprising a spacer group or mixtures thereof.
• the vesicular compositions may comprise one or more other vesicle-forming lipids.
• a vesicle may comprise lipid-spacer derivatives of the invention and other vesicle-forming lipids (co-lipids), preferably in a ratio from 1:200 to 200:1.
• vesicular compositions in form of lipid coated nanoparticles, liposomes, micelles, or other vesicles may be readily prepared from the compounds of the invention using standard conditions known in the art.
• vesicular compositions may be prepared from compounds of the invention optionally in combination with one or more co-lipids including stabilizing lipids.
• the particular stabilizing compounds which are ultimately combined with the present compounds may be selected as desired to optimize the properties of the resulting composition (and are readily identifiable by one skilled in the art without undue experimentation).
• Vesicular compositions of the invention are particularly effective as carriers for the delivery of bioactive agents or as antigen presenting carriers.
• bioactive agent refers to any synthetic or naturally occurring compound (in free form, salt form or solvated or hydrated form) having a biological activity, such as a targeting agent, an antigenic agent, a therapeutic agent or a diagnostic agent, preferably a therapeutic agent or a diagnostic agent.
• antigen-presenting system also termed “antigen display system” as used herein refers to a naturally occurring or synthetic system, which (i) can present at least one antigen (or part thereof) in such a way that the at least one antigen (or part thereof) can be recognized or bound by an immune effector molecule, e.g. a T-cell antigen receptor on the surface of a T cell, or (ii) is capable of presenting at least one antigen (or part thereof) in the form of an antigen-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen (or part thereof) being presented.
• an immune effector molecule e.g. a T-cell antigen receptor on the surface of a T cell
• Micellar vesicular compositions according to the invention may be prepared using any one of a variety of conventional micellar preparatory methods which will be apparent to those skilled in the art. These methods typically involve suspension of the lipid compound in an organic solvent, evaporation of the solvent, resuspension in an aqueous medium, sonication and centrifugation. The foregoing methods, as well as others, are discussed, for example, in Canfield et al., Methods in Enzymology, Vol. 189, pp. 418-422 (1990); El-Gorab et al, Biochem. Biophys. Acta, Vol. 306, pp.
• micellar compositions produced therefrom include lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, myristyltrimethylammonium bromide, (C12-C16)alkyldimethylbenzylammonium chloride, cetylpyridinium bromide and chloride, lauryl sulphate, and the like.
• Other materials for stabilizing the micellar compositions, in addition to those exemplified above, would be apparent to one skilled in the art based on the present disclosure.
• Liposomal vesicular compositions may comprise one or more non-derivatized compounds and/or one or more derivatized compounds (carrying a spacer group) optionally in combination with one or more further co-lipids and/or one or more stabilizing compounds.
• the present compound(s) (optionally in combination with the colipids) may be in form of a monolayer or bilayer. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric.
• the present compounds may be used to form a unilamellar liposome (comprised of one monolayer or bilayer), an oligolamellar liposome (comprised of two or three monolayers or bilayers) or a multilamellar liposome (comprised of more than three monolayers or bilayers).
• compositions of the invention include preferably cationic lipids, phosphatidylcholine (PC), phosphatidyl-DL-glycerol (PG), L- ⁇ -phosphtidylethanolamine (PE), cholesterol, cholesteryl hemisuccinate tri salt (CHEMS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)
• PC phosphatidylcholine
• PG phosphatidyl-DL-glycerol
• PE L- ⁇ -phosphtidylethanolamine
• CHEMS cholesteryl hemisuccinate tri salt
• DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
• the amount of stabilizing material, such as, for example, additional amphipathic compound, which is combined with the present compounds may vary depending upon a variety of factors, including the specific structure of the present compound(s) of the invention selected, the specific stabilizing material(s) selected, the particular use for which it is being employed, the mode of delivery, and the like.
• the amount of stabilizing material to be combined with the present compounds and the ratio of stabilizing material to present compound will vary and is readily determinable by one skilled in the art based on the present disclosure. Typically ratios higher than about 4:1, 3:1 or 2:1, of present compound to stabilizing lipid, are preferred.
• the liposomes may be prepared using any one of a variety of conventional liposome preparatory techniques which will be apparent to those skilled in the art. These techniques include ethanol injection, thin film technique, homogenizing, solvent dialysis, forced hydration, reverse phase evaporation, microemulsification and simple freeze-thawing, Using e.g. conventional microemulsification equipment.
• Additional methods for the preparation of liposomal vesicular compositions of the invention from the compounds of the present invention include, for example, sonication, chelate dialysis, homogenization, solvent infusion, spontaneous formation, solvent vaporization, controlled detergent dialysis, and others, each involving the preparation of liposomes in various ways.
• methods which involve ethanol injection, thin film technique, homogenizing and extrusion are preferred in connection with the preparation of liposomal compositions of the invention from the compounds of the present invention.
• the size of the liposomes can be adjusted, if desired, by a variety of techniques, including extrusion, filtration, sonication and homogenization. Other methods for adjusting the size of the liposomes and for modulating the resultant liposomal biodistribution and clearance of the liposomes would be apparent to one skilled in the art based on the present disclosure.
• the size of the liposomes is adjusted by extrusion under pressure through pores of a defined size.
• the liposomal compositions of the invention may be of any size, preferably less than about 200 nanometer (nm) in outside diameter.
• Nanoparticulate vesicular compositions or nanoparticles are typically small particles having typically a diameter of less than 1 micron, preferably in the range of about 25-1000 nm, more preferably in the range of about 50-300 nm, most preferably in the range of about 60-200 nm.
• a nanoparticle can have any shape and any morphology. Examples of nanoparticles include nanopowders, nanoclusters, nanocrystals, nanospheres, nanofibers, and other geometries.
• a nanopolymer refers to a polymer that upon polymerization assembles to form a nanoparticle, such as, e.g., a nanorod, nanofiber, or nanosphere.
• a nanosphere refers to a type of nanoparticle that is approximately spherical in shape and may have a hollow core or a solid core.
• nanoparticles have a matrix core structure which may be formed using all types of materials and structures, including inorganic materials, such as metals, and organic materials, such as polymers including physiologically acceptable polymers.
• Non-limiting examples of such polymers include, for example, polyesters (such as poly(lactic acid), poly(L-lysine), poly(glycolic acid) and poly(lactic-co-glycolic acid)), poly(lactic acid-co-lysine), poly(lactic acid-graft-lysine), polyanhydrides (such as poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), copolymers of these monomers and the like), poly(anhydride-co-imides), poly(amides), poly(orthoesters), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate
• the nanoparticles may also include hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan, chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc. as well as their derivatives, co-polymers, and mixtures thereof.
• HPC hydroxypropyl cellulose
• NIPA N-isopropylacrylamide
• PVA polyethylene glycol
• PVA polyvinyl alcohol
• polyethylenimine chitosan
• chitin dextran sulfate
• heparin chondroitin sulfate
• chondroitin sulfate gelatin, etc.
• a non-limiting method for making nanoparticles is described e.g. in U.S. Publication 2003/0138490.
• the core material may be selected from metals, alloys, metalloids, metal compounds such as metal oxides, inorganic compounds, and carbon-based materials, in particular carbon nanotubes, one-dimensional nanoparticles of fullerene C 6 o, and three-dimensional nanoparticles of fullerene C 70 .
• metals include, but are not limited to, noble or a platinum metal such as Ag, Au, Pd, Pt, Rh, Ir, Ru, and Os, transition metals such as Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Zr, Nb, Mo, Ta, W, Re, and main group metals such as Al, Ga, In, Si, Ge, Sn, Sb, Bi, Te. It will be appreciated that some main group metals, in particular Si and Ge, are also commonly referred to as metalloids.
• alloys include, but are not limited to, alloys of noble or platinum metal and transition metals, in particular alloys of silver and transition metals such as Ag/Ni, Ag/Cu, Ag/Co, and platinum and transition metals such as Pt/Cu, or noble or platinum alloys such as Ru/Pt.
• inorganic compounds include, but are not limited, to SiO 2 , metal compounds, in particular metal oxides such as TiO 2 and iron oxides.
• Nanoparticles optionally include a functional group such as, for example, a carboxyl, sulhydryl, hydroxyl, or amino group, for covalently linking other compounds, such as linkers, to the surface of a nanoparticle.
• a functional group such as, for example, a carboxyl, sulhydryl, hydroxyl, or amino group
• compounds, such as linkers may be associated to a nanoparticle through other intermolecular forces such as Van-der-Waals forces, ionic interactions, hydrophobic interactions.
• the nanoparticles can be associated with a bioactive agent (e.g., entangled, embedded, incorporated, encapsulated, bound to the surface, or otherwise associated with the nanoparticle).
• a bioactive agent e.g., entangled, embedded, incorporated, encapsulated, bound to the surface, or otherwise associated with the nanoparticle.
• a bioactive agent is associated to a nanoparticle through a compound of the invention acting as a linker between bioactive agent and nanoparticle.
• Nanoparticles may also be grouped together (optionally with a dispersing agent) to form a nanocluster.
• the independent formulation of each nanoparticle type before cluster formation and a special arrangement of nanoparticles within the cluster can allow controlling the duration and concentration of a bioactive ingredient.
• one or more non-derivatized or derivatized compounds of the invention may be incorporated into, attached to or adsorbed to a nanoparticle.
• lipid coated nanoparticles may be formed from nanosized core particles and one or more compounds of the present invention and optionally one or more co-lipids.
• the lipids may be in the form of a monolayer or a bilayer. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric.
• Coating of the nanoparticles is preferably carried out in a solution comprising the compounds of the invention and by allowing sufficient time to allow the compounds to coat the nanoparticles (using techniques known in the art, see e.g. Journal of Controlled Release, Vol 137(1), 69-77, 2009).
• the lipids may be in the form of a monolayer or a bilayer. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric.
• vaporization methods e.g., free jet expansion, laser vaporization, spark erosion, electro explosion and chemical vapor deposition
• physical methods involving mechanical attrition e.g., the pearlmilling technology, Elan Nanosystems, Ireland
• any of the present compounds and vesicular compositions containing the compounds of the invention, with or without bioactive agents may be lyophilized for storage, and reconstituted in, for example, an aqueous medium (such as sterile water or phosphate buffered solution, or aqueous saline solution), preferably under vigorous agitation.
• aqueous medium such as sterile water or phosphate buffered solution, or aqueous saline solution
• additives may be included to prevent agglutination or fusion of the lipids as a result of lyophilisation.
• Useful additives include, without limitation, sorbitol, mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone and poly(ethylene glycol), for example, PEG 400.
• the present compounds and in particular the liposomal compositions of the present invention are particularly suitable for use as carriers for a targeted delivery of bioactive agents or for use as antigen display systems.
• the compounds of the present invention are particularly applicable for use in vitro and/or in vivo in methods for the treatment of diseases, for which a targeted delivery of one or more specific biologically active agents is desirable or required, as well as for use in methods in vitro/in vivo diagnostic applications.
• the present invention relates to a kit comprised of a container that is compartmentalized for holding the various elements of the kit.
• One compartment may contain a predetermined quantity of either a compound of the present invention or a vesicular composition thereof.
• these may be with or without a pH buffer to adjust the composition pH to physiological range of about 7 to about 8, or else in lyophilized or freeze dried form for reconstitution at the time of use.
• Also included within the kit will be other reagents and instructions for use.
• Cholesterol and POPC are purchased from Avanti Polar Lipids (Alabaster, Ala.). All Protected amino acids are obtained from Novabiochem. Diphenyldiazomethane resin D-2230 is obtained from Bachem AG. All other chemicals and solvents are A.R. grade or above.
• 2,3-Bis(tetradecyloxy)propan-1-amine is synthesized according to Kokotos et al. Chemistry-A European Journal, 2000, vol. 6, #22, 4211-4217.
• bis(3-((Z)-octadec-9-enyloxy)propyl)amine is obtained from oleyl methanesulfonate and bis(3-hydroxypropyl)amine (see MaGee et al., J. Journal of Organic Chemistry, 2000, vol. 65, #24, 8367-8371).
• the resin is treated with 125 ⁇ l acetic acid (0.5 eq., 2.2 mmol) in 30 ml DCM for 15 minutes and washed afterwards three times alternating with 30 ml dimethylformamide and isopropanol.
• the resin is washed twice with diisopropyl ether and dried over night in vacuo. 6.7 g of the desired product are obtained (>100% of theory, yield in theory 6.5 g).
• the loading of the resin is determined to 0.49 mmol/g by UV measurement of the Fmoc cleavage product at 304 nm (maximum loading in theory 0.51 mmol/g).
• H-Glu-OtBu-NH-PEG11-Glu(DMA)-diphenylmethyl resin is obtained through conventional solid phase synthesis by the following reaction sequence:
• the desired compound can be obtained by cleavage from the resin e.g. by treatment with trifluaroacetic acid and triisopropylsilane.

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